APPLICATIONS

Background: mTORC1 kinase complex is a critical component in the regulation of cell growth (1,2). Its activity is modulated by energy levels, growth factors, and amino acids (3,4). The four related GTPases, RagA, RagB, RagC, and RagD, have been shown to interact with raptor in mTORC1 (1,2). These interactions are both necessary and sufficient for mTORC1 activation in response to amino acid signals (1,2). A protein complex consisting of LAMTOR1/C11orf59, LAMTOR2/ROBLD3, and LAMTOR3/MAPKSP1 has been identified to interact with and recruit the four Rag GTPases to the surface of lysosomes (5).

APPLICATIONS

Background: Parkinson’s disease (PD), the second most common neurodegenerative disease after Alzheimer’s, is a progressive movement disorder characterized by rigidity, tremors, and postural instability. The pathological hallmarks of PD are progressive loss of dopaminergic neurons in the substantia nigra of the ventral midbrain and the presence of intracellular Lewy bodies (protein aggregates of α-synuclein, ubiquitin, and other components) in surviving neurons of the brain stem (1). Research studies have shown various genes and loci are genetically linked to PD including α-synuclein/PARK1 and 4, parkin/PARK2, UCH-L1/PARK5, PINK1/PARK6, DJ-1/PARK7, LRRK2/PARK8, synphilin-1, and NR4A2 (2).Leucine-rich repeat kinase 2 (LRRK2) contains amino-terminal leucine-rich repeats (LRR), a Ras-like small GTP binding protein-like (ROC) domain, an MLK protein kinase domain, and a carboxy-terminal WD40 repeat domain. Research studies have linked at least 20 LRRK2 mutations to PD, with the G2019S mutation being the most prevalent (3). The G2019S mutation causes increased LRRK2 kinase activity, which induces a progressive reduction in neurite length that leads to progressive neurite loss and decreased neuronal survival (4). Researchers are currently testing the MLK inhibitor CEP-1347 in PD clinical trials, indicating the potential value of LRRK2 as a therapeutic target for treatment of PD (5).

APPLICATIONS

Background: Parkinson’s disease (PD), the second most common neurodegenerative disease after Alzheimer’s, is a progressive movement disorder characterized by rigidity, tremors, and postural instability. The pathological hallmarks of PD are progressive loss of dopaminergic neurons in the substantia nigra of the ventral midbrain and the presence of intracellular Lewy bodies (protein aggregates of α-synuclein, ubiquitin, and other components) in surviving neurons of the brain stem (1). Research studies have shown various genes and loci are genetically linked to PD including α-synuclein/PARK1 and 4, parkin/PARK2, UCH-L1/PARK5, PINK1/PARK6, DJ-1/PARK7, LRRK2/PARK8, synphilin-1, and NR4A2 (2).Leucine-rich repeat kinase 2 (LRRK2) contains amino-terminal leucine-rich repeats (LRR), a Ras-like small GTP binding protein-like (ROC) domain, an MLK protein kinase domain, and a carboxy-terminal WD40 repeat domain. Research studies have linked at least 20 LRRK2 mutations to PD, with the G2019S mutation being the most prevalent (3). The G2019S mutation causes increased LRRK2 kinase activity, which induces a progressive reduction in neurite length that leads to progressive neurite loss and decreased neuronal survival (4). Researchers are currently testing the MLK inhibitor CEP-1347 in PD clinical trials, indicating the potential value of LRRK2 as a therapeutic target for treatment of PD (5).

This Cell Signaling Technology antibody is conjugated to biotin under optimal conditions. The biotinylated antibody is expected to exhibit the same species cross-reactivity as the unconjugated LRRK2 (D18E12) Rabbit mAb #13046.

APPLICATIONS

Background: Parkinson’s disease (PD), the second most common neurodegenerative disease after Alzheimer’s, is a progressive movement disorder characterized by rigidity, tremors, and postural instability. The pathological hallmarks of PD are progressive loss of dopaminergic neurons in the substantia nigra of the ventral midbrain and the presence of intracellular Lewy bodies (protein aggregates of α-synuclein, ubiquitin, and other components) in surviving neurons of the brain stem (1). Research studies have shown various genes and loci are genetically linked to PD including α-synuclein/PARK1 and 4, parkin/PARK2, UCH-L1/PARK5, PINK1/PARK6, DJ-1/PARK7, LRRK2/PARK8, synphilin-1, and NR4A2 (2).Leucine-rich repeat kinase 2 (LRRK2) contains amino-terminal leucine-rich repeats (LRR), a Ras-like small GTP binding protein-like (ROC) domain, an MLK protein kinase domain, and a carboxy-terminal WD40 repeat domain. Research studies have linked at least 20 LRRK2 mutations to PD, with the G2019S mutation being the most prevalent (3). The G2019S mutation causes increased LRRK2 kinase activity, which induces a progressive reduction in neurite length that leads to progressive neurite loss and decreased neuronal survival (4). Researchers are currently testing the MLK inhibitor CEP-1347 in PD clinical trials, indicating the potential value of LRRK2 as a therapeutic target for treatment of PD (5).

APPLICATIONS

Background: Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of proteins activated in response to nutrient deprivation and in neurodegenerative conditions (1). One of the proteins critical to this process is Beclin-1, the mammalian orthologue of the yeast autophagy protein Apg6/Vps30 (2). Beclin-1 can complement defects in yeast autophagy caused by loss of Apg6 and can also stimulate autophagy when overexpressed in mammalian cells (3). Mammalian Beclin-1 was originally isolated in a yeast two-hybrid screen for Bcl-2 interacting proteins and has been shown to interact with Bcl-2 and Bcl-xL, but not with Bax or Bak (4). While Beclin-1 is generally ubiquitously expressed, research studies have shown it is monoallelically deleted in 40-75% of sporadic human breast and ovarian cancers (5). Beclin-1 is localized within cytoplasmic structures including the mitochondria, although overexpression of Beclin-1 reveals some nuclear staining and CRM1-dependent nuclear export (6). Investigators have demonstrated that Beclin-1-/- mice die early in embryogenesis and Beclin-1-/+ mice have a high incidence of spontaneous tumors. Stem cells from the null mice demonstrate an altered autophagic response, although responses to apoptosis appeared normal (7). Researchers have also found that overexpression of Beclin-1 in virally infected neurons in vivo resulted in significant protection against Sindbis virus-induced disease and neuronal apoptosis (4).

APPLICATIONS

Background: Autophagy is a catabolic process for the autophagosomic-lysosomal degradation of proteins activated in response to nutrient deprivation and in neurodegenerative conditions (1). One of the proteins critical to this process is Beclin-1, the mammalian orthologue of the yeast autophagy protein Apg6/Vps30 (2). Beclin-1 can complement defects in yeast autophagy caused by loss of Apg6 and can also stimulate autophagy when overexpressed in mammalian cells (3). Mammalian Beclin-1 was originally isolated in a yeast two-hybrid screen for Bcl-2 interacting proteins and has been shown to interact with Bcl-2 and Bcl-xL, but not with Bax or Bak (4). While Beclin-1 is generally ubiquitously expressed, research studies have shown it is monoallelically deleted in 40-75% of sporadic human breast and ovarian cancers (5). Beclin-1 is localized within cytoplasmic structures including the mitochondria, although overexpression of Beclin-1 reveals some nuclear staining and CRM1-dependent nuclear export (6). Investigators have demonstrated that Beclin-1-/- mice die early in embryogenesis and Beclin-1-/+ mice have a high incidence of spontaneous tumors. Stem cells from the null mice demonstrate an altered autophagic response, although responses to apoptosis appeared normal (7). Researchers have also found that overexpression of Beclin-1 in virally infected neurons in vivo resulted in significant protection against Sindbis virus-induced disease and neuronal apoptosis (4).

APPLICATIONS

Background: Neural precursor expressed, developmentally down-regulated protein 4 (NEDD4) was originally identified as a gene that is highly expressed in the early mouse embryonic central nervous system (1). Subsequently, a family of NEDD4-like proteins have been defined that includes seven members in humans (2). NEDD4 and NEDD4-like (NEDD4L) proteins contain multiple functional domains including a calcium-dependent phospholipid and membrane binding domain (C2 domain), two to four protein binding domains (WW domains), and an E3 ubiquitin-protein ligase domain (HECT domain). NEDD4 and NEDD4L have been shown to downregulate both neuronal voltage-gated Na+ channels (NaVs) and epithelial Na+ channels (ENaCs) in response to increased intracellular Na+ concentrations (3,4). The WW domains of NEDD4 bind to PY motifs (amino acid sequence PPXY) found in multiple NaV and ENaC proteins; ubiquitination of these proteins is mediated by the HECT domain of NEDD4 and results in their internalization and removal from the plasma membrane. Research studies have shown that mutation of the PY motifs in ENaC proteins is associated with Liddle's syndrome, an autosomal dominant form of hypertension (5). In addition to targeting sodium channels, NEDD4L has also been shown to negatively regulate TGF-β signaling by targeting Smad2 for degradation (6). Mouse and human NEDD4 are rapidly cleaved by caspase proteins during apoptosis, although the significance of this cleavage is not clear (7).

APPLICATIONS

Background: Neural precursor expressed, developmentally down-regulated protein 4 (NEDD4) was originally identified as a gene that is highly expressed in the early mouse embryonic central nervous system (1). Subsequently, a family of NEDD4-like proteins have been defined that includes seven members in humans (2). NEDD4 and NEDD4-like (NEDD4L) proteins contain multiple functional domains including a calcium-dependent phospholipid and membrane binding domain (C2 domain), two to four protein binding domains (WW domains), and an E3 ubiquitin-protein ligase domain (HECT domain). NEDD4 and NEDD4L have been shown to downregulate both neuronal voltage-gated Na+ channels (NaVs) and epithelial Na+ channels (ENaCs) in response to increased intracellular Na+ concentrations (3,4). The WW domains of NEDD4 bind to PY motifs (amino acid sequence PPXY) found in multiple NaV and ENaC proteins; ubiquitination of these proteins is mediated by the HECT domain of NEDD4 and results in their internalization and removal from the plasma membrane. Research studies have shown that mutation of the PY motifs in ENaC proteins is associated with Liddle's syndrome, an autosomal dominant form of hypertension (5). In addition to targeting sodium channels, NEDD4L has also been shown to negatively regulate TGF-β signaling by targeting Smad2 for degradation (6). Mouse and human NEDD4 are rapidly cleaved by caspase proteins during apoptosis, although the significance of this cleavage is not clear (7).

APPLICATIONS

Background: HDAC6 is a class II histone deacetylase enzyme localized to the cytoplasm and associated with the microtubule network (1). It is involved in the regulation of many cellular processes, including cell migration, immune synapse formation, viral infection, and degradation of misfolded proteins (1). HDAC6 contains two tandem catalytic domains that facilitate the deacetylation of multiple protein substrates, including histones and non-histone proteins such as tubulin, cortactin, and HSP90. Despite the ability to deacetylate histone proteins in vitro, there is no evidence for HDAC6-mediated deacetylation of histones in vivo (2,3). The acetylation/deacetylation of tubulin on Lys40 regulates binding and motility of the kinesin-1 motor protein and subsequent transport of cargo proteins such as JNK-interacting protein 1 (JIP1) (4). The acetylation/deacetylation of cortactin regulates cell motility by modulating the binding of cortactin to F-actin (5). Acetylation/deacetylation of HSP90 modulates chaperone complex activity by regulating the binding of an essential cochaperone protein, p23 (6,7). In addition to its role as a protein deacetylase, HDAC6 functions as a component of the aggresome, a proteinaceous inclusion body that forms in response to an accumulation of misfolded or partially denatured proteins (8). Formation of the aggresome is a protective response that sequesters cytotoxic protein aggregates for eventual autophagic clearance from the cell. HDAC6 contains a zinc finger ubiquitin-binding domain that binds both mono- and poly-ubiquitinated proteins (8). HDAC6 binds to both poly-ubiquitinated misfolded proteins and dynein motors, facilitating the transport of misfolded proteins to the aggresome (9,10). HDAC6 is also required for subsequent recruitment of the autophagic machinery and clearance of aggresomes from the cell (11). Thus, HDAC6 plays a key role in the protection against the deleterious effects of pathological protein aggregation that occurs in various diseases, such as neurodegenerative Huntington’s disease (11).